HIV-1 infection continues to be a pandemic problem. The goal for a cure remains elusive and will require novel therapeutic approaches. One of the recently proposed technologies utilizes the CRISPR bacterial immunity system where the protein Cas9 is targeted by a guide RNA (gRNA) and induces a sequence specific double strand break. Such DNA damage is generally repaired by the error prone non homologous end joining pathway which typically introduces insertions and deletions at the repair junction. This technology has been proposed to delete the HIV-1 provirus. However, the full effects of CRISPR targeting of the HIV-1 genome are unknown. Only small subsets of gRNAs have been tested for reduction of HIV-1 replication. The time required for the virus to generate resistance mutations has not been revealed and the relative efficiency of gRNAs throughout the HIV-1 genome has not been systematically explored. This proposal contains two highly focused Specific Aims: 1.) Develop a whole genome HIV-1 CRISPR/Cas9 gRNA library for use in an HIV-1 screen and 2.) Map the HIV-1 whole genome mutation profile in response to CRISPR/Cas9 selective pressure. We will generate a library of CRISPR gRNAs targeting the entire HIV-1 subtype B genome, excluding the variable regions of the env gene. This library will be characterized for both the relative reduction in HIV-1 replication and the time to develop resistance. Resistant strains will be analyzed by NextGen sequencing to reveal the nature of the mutations and a heat map of HIV-1 mutability in response to selective pressure applied evenly across the viral genome. Ultimately, this exploratory grant will provide a quantitative and adaptable platform for probing the evolution of HIV-1 in response to selective pressure. These data will yield significant insight into the use of CRISPR as a novel therapeutic targeting the HIV-1 provirus.